Previous research from this laboratory has demonstrated CO(2)-fixing and H(2)-uptake capacities of certain strains of Rhizobium japonicum. In this report we have shown that SR, a H(2)-uptake-positive (Hup(+)) strain of R. japonicum, is capable of autotrophic growth with H(2) as the energy source. Growth occurred on mineral salts/vitamins/Noble agar, mineral salts/vitamins liquid medium (0.27 mug of C as vitamins per ml), and in mineral salts liquid medium with no added vitamins when cultures were provided with NH(4)Cl and incubated in an atmosphere containing H(2), CO(2), O(2), and N(2). Little or no growth occurred when either H(2) or CO(2) was omitted from the atmosphere or when the culture was inoculated with SR3, a Hup(-) mutant of SR. Growth was measured by protein synthesis, fixed organic carbon, and increase in cell number in liquid cultures. The organism that grew autotrophically was verified as R. japonicum by (i) apparent purity on streak plates; (ii) retention of the double antibiotic resistance markers; and (iii) its capability to nodulate soybeans. H(2)- and CO(2)-supported growth was demonstrated for three additional Hup(+) wild-type R. japonicum strains (USDA 136, 3I1b 6, and 3I1b 143), while three Hup(-) wild-type strains (USDA 120, 3I1b 144, and USDA 117) were incapable of growth on the Noble agar medium containing mineral salts/vitamins in the H(2)/CO(2)/O(2)/N(2) atmosphere. This demonstrated capability of Hup(+)R. japonicum strains to grow autotrophically requires revision of current concepts regarding conditions for survival and competition of these bacteria in the soil and their relationships to other microorganisms.